Real time analysis of β(2)-adrenoceptor-mediated signaling kinetics in human primary airway smooth muscle cells reveals both ligand and dose dependent differences

Abstract

Background: β2-adrenoceptor agonists elicit bronchodilator responses by binding to β2-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β2-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP.

Methods: Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β2-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used.

Results: Temporal analysis revealed that in hASM cells the β2-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response.

Conclusions: We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β2-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β2-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo.

Figure 1

Isoproterenol-induced changes in cyclic AMP activity in a single hASM cell imaged via confocal microscopy using the altered emission profile of CFP-Epac(dDEP,CD)-VENUS as a readout. CFP-Epac(dDEP,CD)-VENUS expression 48 hours post-transfection is shown in panel A (CFP excited with 440 nm and 480 nm CFP emission fluorescence recorded). Panel B shows uncorrected FRET in the same cell under basal conditions (CFP excited with 440 nm and 535 nm FRET emission fluorescence recorded). Panel C is a representative trace of the altered emission ratio (essentially a change in FRET) in real time in response to 10 μM isoproterenol (+ Iso).

Figure 2

Cyclic AMP activation induced by a range of doses of isoproterenol in hASM cells using (A) CFP-Epac(dDEP,CD)-VENUS activation or (B) ³H-cyclic AMP formation as a readout. (A) For Epac-based studies single hASM cells expressing CFP-Epac(dDEP,CD)-VENUS were excited at 440 nm and the emission ratio (470/535) changes collected in real time. The maximal ratio was recorded and plotted. Data are expressed as fold over basal. Each data point represents the mean (± SEM) of 3-9 separate experiments. (B) For studies investigating ³H-cyclic AMP formation, monolayers of hASM cells in 24 well plates were labelled with ³H-adenine for 2 hours and then exposed to the appropriate concentrations of isoproterenol for 5 minutes. The reaction was terminated by addition of hydrochloric acid and total ³H-labelled cyclic AMP was collected via column-based separation and quantified by scintillation counting [13]. Data are expressed as fold over basal. Each data point represents the mean (± SEM) of 3-6 experiments.

Figure 3

Cyclic AMP activation induced by single doses of a range of β₂-adrenoceptor agonists in hASM cells using (A) CFP-Epac(dDEP,CD)-VENUS activation or (B) ³H-cyclic AMP formation as a readout. β₂-adrenoceptor agonists studied were isoproterenol (10 μM), salbutamol (1 μM), salmeterol (100 nM), indacaterol (1 μM) and formoterol (1 μM). (A) For FRET-based studies single hASM cells expressing CFP-Epac(dDEP,CD)-VENUS were excited at 440 nm and the emission ratio (470/535) changes collected in real time. The maximum ratio observed was recorded and plotted. Data are expressed as fold over basal. Each data point represents the mean (± SEM) of 6-16 separate experiments. **denotes P < 0.01. (B) For studies investigating ³H-cyclic AMP formation, monolayers of hASM cells in 24 well plates were labelled with ³H-adenine for 2 hours and then exposed to the appropriate concentrations of β₂-adrenoceptor agonist for 5 minutes. The reaction was terminated by addition of hydrochloric acid and total ³H-labelled cyclic AMP was collected via column-based separation and quantified by scintillation counting [13]. Data are expressed as fold over basal. Each data point represents the mean (± SEM) of 3-9 experiments. * denotes P < 0.05, **denotes P < 0.01.

Figure 4

Time taken for a range of β₂-adrenoceptor agonists to (A) initiate an increase in CFP-Epac(dDEP,CD)-VENUS activation and (B) to induce a maximal response c.f. initiation. The time between response initiation and maximal response is shown in C. The labelled trace in D depicts how these data were obtained from real time traces. The β₂-adrenoceptor agonists studied were isoproterenol (10 μM), salbutamol (1 μM), salmeterol (100 nM), indacaterol (1 μM) and formoterol (1 μM). Single hASM cells expressing CFP-Epac(dDEP,CD)-VENUS were excited at 440 nm and the emission ratio (470/535) changes collected in real time. Each data point represents the mean (± SEM) of 6-19 experiments. * denotes P < 0.05, ***denotes P < 0.001.

Figure 5

Re-analysis of data shown in figure 4 concentrating on the response observed in the first 30 seconds after initiation i.e. at a timepoint whereby probe saturation has not occurred. Data was analysed via (A) Area Under the Curve (AUC). B outlines the region utilised for these additional analyses. Each data point represents the mean (± SEM) of 6-19 experiments.* denotes P < 0.05, ** denotes P < 0.01.

Figure 6

Time taken for a range of concentrations of the β₂-adrenoceptor agonist isoproterenol (10^-8M-10^-5M) to initiate an increase in CFP-Epac(dDEP,CD)-VENUS activation. These data were obtained from real time traces comparable to those depicted in Figure 4D. Single hASM cells expressing CFP-Epac(dDEP,CD)-VENUS were excited at 440 nm and the emission ratio (470/535) changes collected in real time. Each data point represents the mean (± SEM) of 4-19 experiments. * denotes P < 0.05.